Method for generating a three-dimensional nucleic acid containing matrix

ABSTRACT

Methods of making a three-dimensional matrix of nucleic acids within a cell is provided.

RELATED APPLICATION DATA

This application is a continuation application which claims priority toU.S. patent application Ser. No. 14/774,282, filed on Sep. 10, 2015,which is a National Stage Application under 35 U.S.C. 371 of co-pendingPCT Application No. PCT/US2014/018580 designating the United States andfiled Feb. 26, 2014; which claims the benefit of U.S. ProvisionalApplication No. 61/777,383 and filed Mar. 12, 2013 each of which arehereby incorporated by reference in their entireties.

STATEMENT OF GOVERNMENT INTERESTS

This invention was made with Government support under grant numberRC2HL102815 awarded by NHLBI and 1P50HG005550 awarded by NHGRI. TheGovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to methods of making a three-dimensionalmatrix of nucleic acids and amplifying, detecting and sequencing suchnucleic acids within the matrix.

BACKGROUND OF THE INVENTION

Since many gene products such as RNA and proteins are enriched inregions where they function, their location provides an important clueto their function. This property has been used for in situ fluorescenthybridization, immunohistochemistry and tissue-specific reporter assaysin numerous areas of biological research.

Current methods involve extracting nucleic acid molecules from theirnative environment or making synthetic nucleic acid molecules,amplifying them in solution and placing them on a flat array surface orbeads for gene detecting via hybridization or sequencing, making itimpossible to identify the cellular origin of individual nucleic acids.

SUMMARY

Embodiments of the present invention are directed to methods of making athree dimensional matrix of nucleic acids. Embodiments of the presentinvention are directed to methods of making a three dimensional matrixincluding nucleic acids covalently bound into a matrix or into or to amatrix material. The nucleic acids may be co-polymerized with the matrixmaterial or cross-linked to the matrix material or both. According toone aspect, a plurality of nucleic acid sequences of certain length,such as DNA or RNA sequences are part of a three-dimensional copolymer.The nucleic acids may then be amplified and sequenced in situ, i.e.within the matrix. The three-dimensional matrix of nucleic acidsprovides, in a certain aspect, an information storage medium where thenucleic acids, i.e. a sequence of one or more nucleotides, representstored information which can be read within the three-dimensionalmatrix. According to one aspect, nucleic acids such as DNA or RNAsequences of given length are covalently attached to a matrix materialto preserve their spatial orientation in the x, y and z axes within thematrix. It is to be understood that the three dimensional matrix mayinclude a matrix material and that the term copolymer, matrix and matrixmaterial may be used interchangeably.

According to one aspect, methods described herein are directed toimmobilizing naturally occurring nucleic acids within their nativeenvironment, such as within a cell or within a tissue sample. The threedimensional nucleic acid matrix can be generated in situ in a cell ortissue sample to preserve the naturally occurring nucleic acid sequencediversity (such as DNA and RNA) and spatial orientation in cells,tissues or any other complex biomaterial. According to this aspect, thelocation of nucleic acids and their relative position is identified as athree dimensional structure, such as within subcellular compartments,within cells, within tissues, as three dimensional nucleic acidassemblies, as three dimensional nucleic acid material, etc. The nucleicacids can be amplified and sequenced, if desired, in situ therebyproviding positional information of the nucleic acids within the cell ortissue.

According to a related aspect, nucleic acids of interest, whethernaturally occurring or synthetic, can be present within a threedimensional matrix material and covalently attached to the threedimensional matrix material such that the relative position of eachnucleic acid is fixed, i.e. immobilized, within the three dimensionalmatrix material. In this manner, a three-dimensional matrix ofcovalently bound nucleic acids of any desired sequence is provided. Eachnucleic acid has its own three dimensional coordinates within the matrixmaterial and each nucleic acid represents information. In this manner, alarge amount of information can be stored in a three dimensional matrix.Individual information-encoding nucleic acids, such as DNA or RNA can beamplified and sequenced in situ, i.e., within the matrix, therebyenabling a large amount of information to be stored and read in asuitable three dimensional material.

According to a further aspect, the nucleic acids can be amplified toproduce amplicons within the three dimensional matrix material. Theamplicons can then be covalently attached to the matrix, for example, bycopolymerization or cross-linking. This results in a structurally stableand chemically stable three dimensional matrix of nucleic acids.According to this aspect, the three dimensional matrix of nucleic acidsallows for prolonged information storage and read-out cycles. Thenucleic acid/amplicon matrix allows for high throughput sequencing of awide ranging array of biological and non-biological samples in threedimensions.

According to certain aspects, a three dimensional nucleic acid matrix isprovided where a plurality of nucleic acid molecules, such as DNA orRNA, amplicons or nucleic acid structural units are immobilized, such asby covalent bonding to the matrix, in a three dimensional space relativeto one another. In this context, the nucleic acid molecules are rigidlyfixed to the extent that they maintain their coordinate position withinthe matrix. It is to be understood that even though a nucleic acidmolecule may be covalently attached to the three dimensional matrixmaterial, the nucleic acid molecule itself may be capable of movementthough bound to the matrix, such as for example, when a nucleic acidsequence is bound to the matrix at a single location on the nucleicacid.

According to one aspect, the three dimensional matrix including nucleicacids is porous. According to one aspect, the three dimensional matrixincluding nucleic acids is porous to the extent that reagents typicallyused in amplification methods can diffuse or otherwise move through thematrix to contact nucleic acids and thereby amplify nucleic acids undersuitable conditions.

According to one aspect, the three dimensional matrix material ischemically inert and thermally stable to allow for various reactionconditions and reaction temperatures. According to this aspect, thethree dimensional matrix material is chemically inert and thermallystable to conditions used in amplification and sequencing methods knownto those of skill in the art.

According to one aspect, the three dimensional matrix material isoptically transparent. According to one aspect, the three dimensionalmatrix material is optically transparent to allow for three dimensionalimaging techniques known to those of skill in the art.

According to one aspect, the nucleic acids are amplified to an extent toproduce sufficient levels of amplicons for three dimensional imaging.For example, the nucleic acids are amplified and include a labelsufficient for a high level of fluorescence compatible with threedimensional imaging.

According to one aspect, the material used to form the matrix iscompatible with a wide range of biological and non-biological specimensin situ so as to avoid extracting the nucleic acid molecules away fromtheir native environment.

According to one aspect, the matrix material may be a semi-solid mediumthat can be made from polyacrylamide, cellulose, alginate, polyamide,cross-linked agarose, cross-linked dextran or cross-linked polyethyleneglycol. In certain aspects, the semi-solid medium has x, y and z axes,and the nucleic acids are present randomly or non-randomly within thethree dimensional matrix.

According to one aspect, the matrix material is porous. Porosity canresult from polymerization and/or crosslinking of molecules used to makethe matrix material. The diffusion property within the gel matrix islargely a function of the pore size. The molecular sieve size is chosento allow for rapid diffusion of enzymes, oligonucleotides, formamide andother buffers used for amplification and sequencing (>50-nm). Themolecular sieve size is also chosen so that large DNA or RNA ampliconsdo not readily diffuse within the matrix (<500-nm). The porosity iscontrolled by changing the cross-linking density, the chain lengths andthe percentage of co-polymerized branching monomers according to methodsknown to those of skill in the art.

In certain aspects, the semi-solid medium can be attached to a solidsupport such as a microscope slide or a flow cell. The solid support canbe attached to the bottom surface of the semi-solid medium.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages of the present inventionwill be more fully understood from the following detailed description ofillustrative embodiments taken in conjunction with the accompanyingdrawings.

FIG. 1 depicts a schematic of nucleic acids at relative positions withina three dimension environment and extraction and placement onto a twodimensional environment, such as a glass slide or flow chamber.

FIG. 2 depicts in schematic the process of creating a matrix of nucleicacids within cells in situ, followed by amplifying the nucleic acids,such as DNA or RNA, in situ, co-polymerizing the amplicons in situ,covalently attaching the amplicons to the matrix material, interrogatingthe amplicons and imaging the amplicons along with a reconstructed 3Dcell image with DNA/RNA amplicons on the order of 10-7 m.

FIG. 3 is an image of a whole mount Drosophilia embryo.

FIG. 4 is an optical section of a fly embryo.

FIG. 5 is an image of a whole mount mouse brain section.

FIG. 6 is an optical section of a mouse brain.

FIG. 7A is a gel image of aminoallyl dUTP after 1 hour of rolling circleamplification and after 4 hours of rolling circle amplification

FIG. 7B is a graph representative of the molar ratio of aminoallyl dUTPto dTTP during amplification.

FIGS. 7C-E depict DNA amplicons with no shear stress (7C), DNA ampliconswith no crosslinking and stretched out from 5 minutes of high shearstress (7D), and DNA amplicons with aminoallyl dUTP cross-linking beingmorphologically preserved after 5 minutes of high shear stress.

FIG. 8A depicts DNA amplicons cross-linked in fibroblasts.

FIG. 8B depicts the results of experiments demonstrating structuralintegrity of a DNA amplicon matrix within a cell.

FIG. 8C depicts the results of experiments demonstrating structuralintegrity of a DNA amplicon matrix within a cell after numerous chemicalreactions.

FIG. 9A depicts amplicons within pluripotent stem cells.

FIG. 9B depicts confocal microscope images of cells with amplicons beingsequenced.

FIG. 10 depicts in schematic a process for crosslinking orcopolymerizing circular DNA, amplifying the circular DNA to produceamplicons and then placing the DNA amplicons into an ordered 3D matrixusing a suitable scaffold material with addressable primers that canserve as amplification primers.

DETAILED DESCRIPTION

The present invention provides a three dimensional matrix of a pluralityof nucleic acids. The present invention provides a three dimensionalmatrix including a plurality of nucleic acids bound thereto. Accordingto one aspect, the matrix is a three dimensional nucleic acid-containingpolymer. The nucleic acids may be naturally occurring nucleic acids ornon-naturally occurring nucleic acids, such as nucleic acids that havebeen made using synthetic methods. The nucleic acids in the threedimensional matrix may be ordered or unordered. The nucleic acids in thethree dimensional matrix may be present in their natural spatialrelationship within a cell, tissue or organism. The nucleic acids in thethree dimensional matrix may be present in rows and columns within thethree dimensional matrix.

According to one aspect, the nucleic acids are modified to incorporate afunctional moiety for attachment to the matrix. The functional moietycan be covalently cross-linked, copolymerize with or otherwisenon-covalently bound to the matrix. The functional moiety can react witha cross-linker. The functional moiety can be part of a ligand-ligandbinding pair. dNTP or dUTP can be modified with the functional group, sothat the function moiety is introduced into the DNA duringamplification. A suitable exemplary functional moiety includes an amine,acrydite, alkyne, biotin, azide, and thiol. In the case of crosslinking,the functional moiety is cross-linked to modified dNTP or dUTP or both.Suitable exemplary cross-linker reactive groups include imidoester(DMP), succinimide ester (NHS), maleimide (Sulfo-SMCC), carbodiimide(DCC, EDC) and phenyl azide. Cross-linkers within the scope of thepresent disclosure may include a spacer moiety. Such spacer moieties maybe functionalized. Such spacer moieties may be chemically stable. Suchspacer moieties may be of sufficient length to allow amplification ofthe nucleic acid bound to the matrix. Suitable exemplary spacer moietiesinclude polyethylene glycol, carbon spacers, photo-cleavable spacers andother spacers known to those of skill in the art and the like.

According to one aspect, a matrix-forming material is contacted to aplurality of nucleic acids spatially arrange in three-dimensionsrelative to one another.

Matrix forming materials include polyacrylamide, cellulose, alginate,polyamide, cross-linked agarose, cross-linked dextran or cross-linkedpolyethylene glycol. The matrix forming materials can form a matrix bypolymerization and/or crosslinking of the matrix forming materials usingmethods specific for the matrix forming materials and methods, reagentsand conditions known to those of skill in the art.

According to one aspect, a matrix-forming material can be introducedinto a cell. The cells are fixed with formaldehyde and then immersed inethanol to disrupt the lipid membrane. The matrix forming reagents areadded to the sample and are allowed to permeate throughout the cell. Apolymerization inducing catalyst, UV or functional cross-linkers arethen added to allow the formation of a gel matrix. The un-incorporatedmaterial is washed out and any remaining functionally reactive group isquenched. Exemplary cells include any cell, human or otherwise,including diseased cells or healthy cells. Certain cells include humancells, non-human cells, human stem cells, mouse stem cells, primary celllines, immortalized cell lines, primary and immortalized fibroblasts,HeLa cells and neurons.

According to one aspect, a matrix-forming material can be used toencapsulate a biological sample, such as a tissue sample. Theformalin-fixed embedded tissues on glass slides are incubated withxylene and washed using ethanol to remove the embedding wax. They arethen treated with Proteinase K to permeabilized the tissue. Apolymerization inducing catalyst, UV or functional cross-linkers arethen added to allow the formation of a gel matrix. The un-incorporatedmaterial is washed out and any remaining functionally reactive group isquenched. Exemplary tissue samples include any tissue samples ofinterest whether human or non-human. Such tissue samples include thosefrom skin tissue, muscle tissue, bone tissue, organ tissue and the like.Exemplary tissues include human and mouse brain tissue sections, embryosections, tissue array sections, and whole insect and worm embryos.

The matrix-forming material forms a three dimensional matrix includingthe plurality of nucleic acids. According to one aspect, thematrix-forming material forms a three dimensional matrix including theplurality of nucleic acids while maintaining the spatial relationship ofthe nucleic acids. In this aspect, the plurality of nucleic acids areimmobilized within the matrix material. The plurality of nucleic acidsmay be immobilized within the matrix material by copolymerization of thenucleic acids with the matrix-forming material. The plurality of nucleicacids may also be immobilized within the matrix material by crosslinkingof the nucleic acids to the matrix material or otherwise cross-linkingwith the matrix-forming material. The plurality of nucleic acids mayalso be immobilized within the matrix by covalent attachment or throughligand-protein interaction to the matrix.

According to one aspect, the matrix is porous thereby allowing theintroduction of reagents into the matrix at the site of a nucleic acidfor amplification of the nucleic acid. A porous matrix may be madeaccording to methods known to those of skill in the art. In one example,a polyacrylamide gel matrix is co-polymerized with acrydite-modifiedstreptavidin monomers and biotinylated DNA molecules, using a suitableacrylamide:bis-acrylamide ratio to control the cross-linking density.Additional control over the molecular sieve size and density is achievedby adding additional cross-linkers such as functionalized polyethyleneglycols. According to one aspect, the nucleic acids, which may representindividual bits of information, are readily accessed byoligonucleotides, such as labeled oligonucleotide probes, primers,enzymes and other reagents with rapid kinetics.

According to one aspect, the matrix is sufficiently opticallytransparent or otherwise has optical properties suitable for standardNext Generation sequencing chemistries and deep three dimensionalimaging for high throughput information readout. The Next Generationsequencing chemistries that utilize fluorescence imaging include ABISoLiD (Life Technologies), in which a sequencing primer on a template isligated to a library of fluorescently labeled nonamers with a cleavableterminator. After ligation, the beads are then imaged using four colorchannels (FITC, Cy3, Texas Red and Cy5). The terminator is then cleavedoff leaving a free-end to engage in the next ligation-extension cycle.After all dinucleotide combinations have been determined, the images aremapped to the color code space to determine the specific base calls pertemplate. The workflow is achieved using an automated fluidics andimaging device (i.e. SoLiD 5500 W Genome Analyzer, ABI LifeTechnologies). Another sequencing platform uses sequencing by synthesis,in which a pool of single nucleotide with a cleavable terminator isincorporated using DNA polymerase. After imaging, the terminator iscleaved and the cycle is repeated. The fluorescence images are thenanalyzed to call bases for each DNA amplicons within the flow cell (HiSeq, Illumia).

According to certain aspects, the plurality of nucleic acids may beamplified to produce amplicons by methods known to those of skill in theart. The amplicons may be immobilized within the matrix generally at thelocation of the nucleic acid being amplified, thereby creating alocalized colony of amplicons. The amplicons may be immobilized withinthe matrix by steric factors. The amplicons may also be immobilizedwithin the matrix by covalent or noncovalent bonding. In this manner,the amplicons may be considered to be attached to the matrix. By beingimmobilized to the matrix, such as by covalent bonding or crosslinking,the size and spatial relationship of the original amplicons ismaintained. By being immobilized to the matrix, such as by covalentbonding or crosslinking, the amplicons are resistant to movement orunraveling under mechanical stress.

According to one aspect, the amplicons, such as DNA amplicons, are thencopolymerized and/or covalently attached to the surrounding matrixthereby preserving their spatial relationship and any informationinherent thereto. For example, if the amplicons are those generated fromDNA or RNA within a cell embedded in the matrix, the amplicons can alsobe functionalized to form covalent attachment to the matrix preservingtheir spatial information within the cell thereby providing asubcellular localization distribution pattern.

As used herein, the term “attach” refers to both covalent interactionsand noncovalent interactions. A covalent interaction is a chemicallinkage between two atoms or radicals formed by the sharing of a pair ofelectrons (i.e., a single bond), two pairs of electrons (i.e., a doublebond) or three pairs of electrons (i.e., a triple bond). Covalentinteractions are also known in the art as electron pair interactions orelectron pair bonds. Noncovalent interactions include, but are notlimited to, van der Waals interactions, hydrogen bonds, weak chemicalbonds (i.e., via short-range noncovalent forces), hydrophobicinteractions, ionic bonds and the like. A review of noncovalentinteractions can be found in Alberts et al., in Molecular Biology of theCell, 3d edition, Garland Publishing, 1994, incorporated herein byreference in its entirety for all purposes.

As used herein, the term “nucleic acid” includes the term“oligonucleotide” or “polynucleotide” which includes a plurality ofnucleotides. The term “nucleic acid” is intended to include naturallyoccurring nucleic acids and synthetic nucleic acids. The term “nucleicacid” is intended to include single stranded nucleic acids and doublestranded nucleic acids. The term “nucleic acid” is intended to includeDNA and RNA, whether single stranded or double stranded. Nucleotides ofthe present invention will typically be the naturally-occurringnucleotides such as nucleotides derived from adenosine, guanosine,uridine, cytidine and thymidine. When oligonucleotides are referred toas “double-stranded,” it is understood by those of skill in the art thata pair of oligonucleotides exists in a hydrogen-bonded, helical arraytypically associated with, for example, DNA. In addition to the 100%complementary form of double-stranded oligonucleotides, the term“double-stranded” as used herein is also meant to include those formwhich include such structural features as bulges and loops (see Stryer,Biochemistry, Third Ed. (1988), incorporated herein by reference in itsentirety for all purposes). As used herein, the term “polynucleotide”refers to a strand of nucleic acids that can be a variety of differentsizes. Polynucleotides may be the same size as an oligonucleotide, ormay be two-times, three-times, four-times, five-times, ten-times, orgreater than the size of an oligonucleotide.

Oligonucleotides and/or polynucleotides may be isolated from naturalsources or purchased from commercial sources. Oligonucleotide and/orpolynucleotide sequences may be prepared by any suitable method, e.g.,the phosphoramidite method described by Beaucage and Carruthers ((1981)Tetrahedron Lett. 22: 1859) or the triester method according toMatteucci et al. (1981) J. Am. Chem. Soc. 103:3185), both incorporatedherein by reference in their entirety for all purposes, or by otherchemical methods using either a commercial automated oligonucleotidesynthesizer or high-throughput, high-density array methods describedherein and known in the art (see U.S. Pat. Nos. 5,602,244, 5,574,146,5,554,744, 5,428,148, 5,264,566, 5,141,813, 5,959,463, 4,861,571 and4,659,774, incorporated herein by reference in its entirety for allpurposes). Pre-synthesized oligonucleotides may also be obtainedcommercially from a variety of vendors.

In certain embodiments of the invention oligonucleotides and/orpolynucleotides may be prepared using a variety of microarraytechnologies known in the art. Pre-synthesized oligonucleotide and/orpolynucleotide sequences may be attached to a support or synthesized insitu using light-directed methods, flow channel and spotting methods,inkjet methods, pin-based methods and bead-based methods set forth inthe following references: McGall et al. (1996) Proc. Natl. Acad. Sci.U.S.A. 93:13555; Synthetic DNA Arrays In Genetic Engineering, Vol.20:111, Plenum Press (1998); Duggan et al. (1999) Nat. Genet. S21:10;Microarrays: Making Them and Using Them In Microarray Bioinformatics,Cambridge University Press, 2003; U.S. Patent Application PublicationNos. 2003/0068633 and 2002/0081582; U.S. Pat. Nos. 6,833,450, 6,830,890,6,824,866, 6,800,439, 6,375,903 and 5,700,637; and PCT Application Nos.WO 04/031399, WO 04/031351, WO 04/029586, WO 03/100012, WO 03/066212, WO03/065038, WO 03/064699, WO 03/064027, WO 03/064026, WO 03/046223, WO03/040410 and WO 02/24597; incorporated herein by reference in theirentirety for all purposes.

Nucleic acids may be obtained from libraries, e.g., genomic libraries,cDNA libraries and the like. Examples of methods for the synthesis ofmolecular libraries can be found in the art, for example in: DeWitt etal. (1993) Proc. Natl. Acad. Sci. USA 90:6909; Erb et al. (1994) Proc.Natl. Acad. Sci. USA 91:11422; Zuckermann et al. (1994) J. Med. Chem.37:2678; Cho et al. (1993) Science 261:1303; Carrell et al. (1994)Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem.Int. Ed. Engl. 33:2061; and in Gallop et al. (1994) J. Med. Chem.37:1233, incorporated herein by reference in their entirety for allpurposes.

In certain embodiments, nucleic acids are those found naturally in abiological sample, such as a cell or tissue.

In still other aspects, a matrix is used in conjunction with a solidsupport. For example the matrix can be polymerized in such a way thatone surface of the matrix is attached to a solid support (e.g., a glasssurface), while the other surface of the matrix is exposed or sandwichedbetween two solid supports. According to one aspect, the matrix can becontained within a container.

Solid supports of the invention may be fashioned into a variety ofshapes. In certain embodiments, the solid support is substantiallyplanar. Examples of solid supports include plates such as slides,microtitre plates, flow cells, coverslips, microchips, and the like,containers such as microfuge tubes, test tubes and the like, tubing,sheets, pads, films and the like. Additionally, the solid supports maybe, for example, biological, nonbiological, organic, inorganic, or acombination thereof.

Embodiments of the present invention are further directed to theamplification of nucleic acid sequences within the matrix, i.e. in situ,within the matrix. Methods of amplifying nucleic acids include rollingcircle amplification in situ. In certain aspects, methods of amplifyingnucleic acids involves the use of PCR, such as anchor PCR or RACE PCR,or, alternatively, in a ligation chain reaction (LCR) (see, e.g.,Landegran et al. (1988) Science 241:1077-1080; and Nakazawa et al.(1994) Proc. Natl. Acad. Sci. U.S.A. 91:360-364; incorporated herein byreference in their entirety for all purposes). Alternative amplificationmethods include: self sustained sequence replication (Guatelli et al.(1990) Proc. Natl. Acad. Sci. USA 87:1874, incorporated herein byreference in its entirety for all purposes), transcriptionalamplification system (Kwoh et al. (1989) Proc. Natl. Acad. Sci. US.86:1173, incorporated herein by reference in its entirety for allpurposes), Q-Beta Replicase (Lizardi et al. (1988) BioTechnology 6:1197,incorporated herein by reference in its entirety for all purposes),recursive PCR (Jaffe et al. (2000) J. Biol. Chem. 275:2619; and Williamset al. (2002) J. Biol. Chem. 277:7790; incorporated herein by referencein their entirety for all purposes) or any other nucleic acidamplification method using techniques well known to those of skill inthe art. A variety of amplification methods are described in U.S. Pat.Nos. 6,391,544, 6,365,375, 6,294,323, 6,261,797, 6,124,090 and5,612,199, incorporated herein by reference in their entirety for allpurposes.

Embodiments of the present invention are directed to methods ofamplifying nucleic acids in situ within the matrix by contacting thenucleic acids within the matrix with reagents and under suitablereaction conditions sufficient to amplify the nucleic acids. Accordingto one aspect, the matrix is porous to allow migration of reagents intothe matrix to contact the nucleic acids. In certain aspects,oligonucleotides are amplified by selectively hybridizing anamplification primer to an amplification site at the 3′ end of anoligonucleotide using conventional methods. Amplification primers are 6to 100, and even up to 1,000, nucleotides in length, but typically from10 to 40 nucleotides, although oligonucleotides of different length areof use. Amplification primers may be present in solution to be added tothe matrix or they may be added during formation of the matrix to bepresent therein sufficiently adjacent to nucleic acids to allow forhybridization and amplification.

Typically, selective hybridization occurs when two nucleic acidsequences are substantially complementary, i.e., at least about 65% 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% 99%, 99.1%, 99.2%,99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% complementaryover a stretch of at least 14 to 25 nucleotides. See Kanehisa, M., 1984,Nucleic Acids Res. 12: 203, incorporated herein by reference in itsentirety for all purposes.

Overall, five factors influence the efficiency and selectivity ofhybridization of the primer to a second nucleic acid molecule. Thesefactors, which are (i) primer length, (ii) the nucleotide sequenceand/or composition, (iii) hybridization temperature, (iv) bufferchemistry and (v) the potential for steric hindrance in the region towhich the primer is required to hybridize, are important considerationswhen non-random priming sequences are designed.

There is a positive correlation between primer length and both theefficiency and accuracy with which a primer will anneal to a targetsequence; longer sequences have a higher Tm than do shorter ones, andare less likely to be repeated within a given target sequence, therebycutting down on promiscuous hybridization. Primer sequences with a highG-C content or that comprise palindromic sequences tend toself-hybridize, as do their intended target sites, since unimolecular,rather than bimolecular, hybridization kinetics are generally favored insolution; at the same time, it is important to design a primercontaining sufficient numbers of G-C nucleotide pairings to bind thetarget sequence tightly, since each such pair is bound by three hydrogenbonds, rather than the two that are found when A and T bases pair.Hybridization temperature varies inversely with primer annealingefficiency, as does the concentration of organic solvents, e.g.,formamide, that might be included in a hybridization mixture, whileincreases in salt concentration facilitate binding. Under stringenthybridization conditions, longer probes hybridize more efficiently thando shorter ones, which are sufficient under more permissive conditions.Stringent hybridization conditions typically include salt concentrationsof less than about 1M, more usually less than about 500 mM andpreferably less than about 200 mM. Hybridization temperatures range fromas low as 0° C. to greater than 22° C., greater than about 30° C., and(most often) in excess of about 37° C. Longer fragments may requirehigher hybridization temperatures for specific hybridization. As severalfactors affect the stringency of hybridization, the combination ofparameters is more important than the absolute measure of any one alone.Hybridization conditions are known to those skilled in the art and canbe found in Current Protocols in Molecular Biology, John Wiley & Sons,N.Y. (1989), 6.3.1-6.3.6, incorporated herein by reference in itsentirety for all purposes.

Primers are designed with the above first four considerations in mind.While estimates of the relative merits of numerous sequences are madementally, computer programs have been designed to assist in theevaluation of these several parameters and the optimization of primersequences (see, e.g., Hoover et al. (2002) Nucleic Acids Res. 30:e43,and Rouillard et al. (2004) Nucleic Acids Res. 32:W176, incorporated byreference herein in their entirety for all purposes).

In accordance with certain examples, methods of sequencing nucleic acidin situ within a matrix are provided. General sequencing methods knownin the art, such as sequencing by extension with reversible terminators,fluorescent in situ sequencing (FISSEQ), pyrosequencing, massivelyparallel signature sequencing (MPSS) and the like (described in Shendureet al. (2004) Nat. Rev. 5:335, incorporated herein by reference in itsentirety), are suitable for use with the matrix in which the nucleicacids are present. Reversible termination methods use step-wisesequencing-by-synthesis biochemistry that coupled with reversibletermination and removable fluorescence (Shendure et al. supra ands U.S.Pat. Nos. 5,750,341 and 6,306,597, incorporated herein by reference.FISSEQ is a method whereby DNA is extended by adding a single type offluorescently-labelled nucleotide triphosphate to the reaction, washingaway unincorporated nucleotide, detecting incorporation of thenucleotide by measuring fluorescence, and repeating the cycle. At eachcycle, the fluorescence from previous cycles is bleached or digitallysubtracted or the fluorophore is cleaved from the nucleotide and washedaway. FISSEQ is described further in Mitra et al. (2003) Anal. Biochem.320:55, incorporated herein by reference in its entirety for allpurposes. Pyrosequencing is a method in which the pyrophosphate (PPi)released during each nucleotide incorporation event (i.e., when anucleotide is added to a growing polynucleotide sequence). The PPireleased in the DNA polymerase-catalyzed reaction is detected by ATPsulfurylase and luciferase in a coupled reaction which can be visiblydetected. The added nucleotides are continuously degraded by anucleotide-degrading enzyme. After the first added nucleotide has beendegraded, the next nucleotide can be added. As this procedure isrepeated, longer stretches of the template sequence are deduced.Pyrosequencing is described further in Ronaghi et al. (1998) Science281:363, incorporated herein by reference in its entirety for allpurposes. MPSS utilizes ligation-based DNA sequencing simultaneously onmicrobeads. A mixture of labelled adaptors comprising all possibleoverhangs is annealed to a target sequence of four nucleotides. Thelabel is detected upon successful ligation of an adaptor. A restrictionenzyme is then used to cleave the DNA template to expose the next fourbases. MPSS is described further in Brenner et al. (2000) Nat. Biotech.18:630, incorporated herein by reference in its entirety for allpurposes.

According to certain aspects, the nucleic acids within the matrix can beinterrogated using methods known to those of skill in the art includingfluorescently labeled oligonucleotide/DNA/RNA hybridization, primerextension with labeled ddNTP, sequencing by ligation and sequencing bysynthesis. Ligated circular padlock probes described in Larsson, et al.,(2004), Nat. Methods 1:227-232 can be used to detect multiple sequencetargets in parallel, followed by either sequencing-by-ligation,-synthesis or -hybridization of the barcode sequences in the padlockprobe to identify individual targets.

According to one aspect, methods described herein produce a threedimensional nucleic acid amplicon matrix which is stable, long-lastingand resistant, substantially resistant or partially resistant toenzymatic or chemical degradation. The three dimensional nucleic acidamplicon matrix can be repeatedly interrogated using standard probehybridization and/or fluorescence based sequencing. The threedimensional nucleic acid amplicon matrix can be repeatedly interrogatedwith little or no signal degradation, such as after more than 50 cycles,and with little position shift, such as less than 1 μm per amplicon.

According to one aspect, a plurality of circular DNA molecules arecovalently linked to one another. The circular DNA molecules are thenamplified using methods known to those of skill in the art, such asisothermal enzymatic amplification one example of which is rollingcircle amplification. According to this aspect, the amplicons arelocalized near the circular DNA. According to this aspect, the ampliconsform a shell around the circular DNA or otherwise assemble around thecircular DNA. Each circular DNA may have more than 1000 ampliconssurrounding or otherwise associated therewith. According to this aspect,the amplicons surrounding a particular circular DNA provide a highsignal intensity, due in part to the number of amplicons and/ordetectable labels associated with the amplicons. The amplicons may befunctionalized and cross-linked or otherwise covalently bound togetheraround their associate circular DNA to form a series or network oftightly bound DNA amplicon shells around each circular DNA. The seriesor network of tightly bound DNA amplicon shells around each circular DNAmay be assembled onto a three-dimensional support. According to oneaspect, the series or network of tightly bound DNA amplicon shellsaround each circular DNA may be assembled onto a three-dimensionalsupport producing a three dimensional DNA polymer with defined overallshape, size and amplicon position.

According to one aspect, amplicons are covalently linked without theneed for separate cross-linkers, such asbis-N-succinimidyl-(nonaethylene glycol) ester. An acrydite moiety, suchas a catalyst activated acrydite moiety is introduced at the end of along carbon spacer (i.e., about C6 to about C12) at position 5 of auracil base a representative formula of which is shown below.

In the formula below, R represents the acrydite spacer moiety attachedto the 5 position of the uracil base.

When copolymerized with bis-acrylamide in the presence of a catalyst, apolymerization reaction takes place, encapsulating the circular DNA withthe amplicons and fixing the amplicons in position. The chemically inertnature of the polymerized mixture allows various downstreamapplications. The spacer can be a carbon chain of between about 2carbons to about 200 carbons. The spacer can be polyethylene glycol. Thelength of the spacer can vary from about 30 angstroms to about 100angstroms and can be of various molecular weights. The spacer can bepermanent or reversible, such as by using UV light, enzymes, chemicalcleavage, etc. A three dimensional matrix, such as a polyacrylamide gelmatrix, can be used to embed a variety of biological structurescontaining enzymatically or chemically modified DNA or RNA moleculescontaining an acrydite functional moiety or moieties. The non-nucleicacid component is selectively dissolved using detergents, proteases,organic solvents or denaturants to create a three dimensional matrixthat preserves individual DNA or RNA molecules and their relativespatial location. Examples include embedding cells, healthy and diseasedtissues and tissue sections, small model organisms such as worms andinsects, bacterial colonies or biofilm, environmental samples containingother DNA or RNA containing materials or organisms.

This invention is further illustrated by the following examples, whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication are hereby incorporated by reference in their entirety forall purposes.

Example I Immobilizing, Amplifying and Imaging DNA/RNA Molecules withinCells

Human iPS cells or human primary fibroblasts are grown on a 1.5 coverslip. They are fixed using 4% formaldehyde in PBS for 15 min, followedby three washes of 70% ethanol. The reverse transcription mixturecontaining 1 uM random hexamer or 0.1 uM polydT(18)V primer withadditional adapter sequences (TCTCGGGAACGCTGAAGA), 250 uM dNTP, 40 uMaminoallyl dUTP (Anaspec), 20 U RNase inhibitor and 100 U MMuLV reversetranscriptase (Enzymatics) are then added to the fixed cells andincubated overnight at 37° C. The sample is then washed using PBS, andcross-linked using 100 uM BS(PEG)9 (Thermo-Fisher Scientific) in PBS for1 hour, followed by 1M Tris treatment for 15 min. The circularizationmixture containing 25 U CircLigase (Epicentre), 1 mM MnCl and 1 M Betainis added, and the sample is incubated at 60° C. for 2 hours. Theresidual RNA is degraded using a mixture of RNase cocktail (Roche) andRNase H (Enzymatics) at 37° C. for 1 hour. The RCA primer is thenhybridized to the sample at 60° C. for 15 min and washed. For rollingcircle amplification, 100 U phi29 DNA polymerase (Enzymatics), 250 uMdNTP and 40 uM aminoallyl dNTP are added to the sample and incubated at30° C. overnight. The sample is then washed using PBS, and cross-linkedusing 100 uM BS(PEG)9 in PBS for 1 hour, followed by 1M Tris treatmentfor 15 min. For the DNA amplicon detection, 1 uM fluorescently labeloligonucleotides are diluted in 2×SSC and hybridized to the matrixcontaining the DNA amplicons at 60° C. and washed. Imaging is done usingLeica SP5 scanning confocal microscope using 10×, 20× or 63× objectivesin four color channels (FITC, Cy3, Texas Red and Cy5). The image stackscontaining up to 50 optical sections are then visualized using ImarisBitplane software for three dimensional reconstruction of the DNAamplicons within the sample matrix.

Methods described herein allow one to immobilize, amplify and imagesingle DNA/RNA molecules in a three dimensional space without perturbingthe structure. As shown in FIG. 2, single cells were grown in tissueculture. DNA/RNA was amplified in situ. The DNA/RNA was co-polymerizedinto a matrix material in situ, and individual amplicons wereinterrogated/hybridized with fluorescent oligonucleotides and imaged.When viewed under much higher magnification, individual amplicons can beimaged using confocal microscopy. This allows one to find out wheredifferent DNA/RNA molecules reside, how they are compartmentalized amongdifferent cell types and morphologies and how their representationchanges over time in developing tissues. The similar concept can be usedfor many other specimens in both natural and synthetic materials, aslong as they can be co-polymerized and/or encapsulated by the DNAamplicons.

According to one specific aspect, inside individual mammalian cells, 20to 500K mRNA molecules are distributed throughput the cytoplasm (Islamet al., 2011). According to embodiment, cells are fixed andpermeabilized. Cellular RNA is then converted into cDNA molecules usingdUTP in place or in addition to dTTP. The cDNA molecules containingmodified dUMP residues are then cross-linked to each other andcircularized, forming a three dimensional pseudo-polymer of circularcDNA molecules inside individual cells. Then rolling circleamplification is used to amplify the cDNA network into a DNA ampliconnetwork. This cell-based DNA amplicon network then stores informationabout each transcript's identity, location, variation/mutations, etc.The cell-based DNA amplicon matrix can be read using sequencing byligation (i.e. ABI SoLiD), sequencing by synthesis (i.e. Illumina), orany other proprietary or open sequencing chemistries (see Drmanac etal., 2010; Shendure et al., 2005 herein incorporated by reference intheir entireties). Given the three dimensional nature of the DNAamplicon network, one can use confocal or multi-photon microscopy tosequencing individual amplicons throughout the whole thickness of theamplicon network, enabling one to visualize the cDNA distribution oftranscripts between the apical side and the basal side of the cells asshown in FIG. 2. Given the tight packing density, one can selectivelyread different subpopulations sequentially, reducing the density ofinformation read at any given time and extending over time for betterspatial resolution.

Example II Immobilizing, Amplifying and Imaging DNA/RNA Molecules withina Fly Embryo

Drosophila embryos are fixed using 4% formaldehyde in PBS, followed bymultiple washes of 70% ethanol. The embryos are then mounted on a coverglass using an optically transparent adhesive. The reverse transcriptionmixture containing 1 uM random hexamer or 0.1 uM polydT(18)V primer withadditional adapter sequences (TCTCGGGAACGCTGAAGA), 250 uM dNTP, 40 uMaminoallyl dUTP (Anaspec), 20 U RNase inhibitor and 100 U MMuLV reversetranscriptase (Enzymatics) are then added to the fixed cells andincubated overnight at 37° C. The sample is then washed using PBS, andcross-linked using 100 uM BS(PEG)9 (Thermo-Fisher Scientific) in PBS for1 hour, followed by 1M Tris treatment for 15 min. The circularizationmixture containing 25 U CircLigase (Epicentre), 1 mM MnCl and 1 M Betainis added, and the sample is incubated at 60° C. for 2 hours. Theresidual RNA is degraded using a mixture of RNase cocktail (Roche) andRNase H (Enzymatics) at 37° C. for 1 hour. The RCA primer is thenhybridized to the sample at 60° C. for 15 min and washed. For rollingcircle amplification, 100 U phi29 DNA polymerase (Enzymatics), 250 uMdNTP and 40 uM aminoallyl dNTP are added to the sample and incubated at30° C. overnight. The sample is then washed using PBS, and cross-linkedusing 100 uM BS(PEG)9 in PBS for 1 hour, followed by 1M Tris treatmentfor 15 min. For the DNA amplicon detection, 1 uM fluorescently labeloligonucleotides are diluted in 2×SSC and hybridized to the matrixcontaining the DNA amplicons at 60° C. and washed. Imaging is done usingLeica SP5 scanning confocal microscope using 10×, 20× or 63× objectivesin four color channels (FITC, Cy3, Texas Red and Cy5). The image stacksare then visualized using Imaris Bitplane software for three dimensionalreconstruction of the DNA amplicons within the sample matrix.

As shown in FIG. 2, fly embryos were obtained and DNA/RNA was amplifiedin situ. The DNA/RNA was copolymerized into a matrix material in situ,and individual amplicons were interrogated/hybridized with fluorescentoligonucleotides and imaged. When viewed under much highermagnification, individual amplicons can be imaged using confocalmicroscopy even in these thick biological specimens. This allows one tofind out where different DNA/RNA molecules reside, how they arecompartmentalized among different cell types and morphologies and howtheir representation changes over time in developing tissues. Thesimilar concept can be used for many other specimens in both natural andsynthetic materials, as long as they can be co-polymerized and/orencapsulated by the DNA amplicons.

Example III Immobilizing, Amplifying and Imaging DNA/RNA Moleculeswithin Mouse Brain

A fresh frozen adult mouse brain sections (20-um cryosections) are fixedusing 4% formaldehyde in PBS. It is then treated with 0.4 ug/mlProteinase K for 30 min at room temperature and thoroughly washed using70%, 95% and 100% ethanol. The reverse transcription mixture containing1 uM random hexamer or 0.1 uM polydT(18)V primer with additional adaptersequences (TCTCGGGAACGCTGAAGA), 250 uM dNTP, 40 uM aminoallyl dUTP(Anaspec), 20 U RNase inhibitor and 100 U MMuLV reverse transcriptase(Enzymatics) are then added to the fixed cells and incubated overnightat 37° C. The sample is then washed using PBS, and cross-linked using100 uM BS(PEG)9 (Thermo-Fisher Scientific) in PBS for 1 hour, followedby 1M Tris treatment for 15 min. The circularization mixture containing25 U CircLigase (Epicentre), 1 mM MnCl and 1 M Betain is added, and thesample is incubated at 60° C. for 2 hours. The residual RNA is degradedusing a mixture of RNase cocktail (Roche) and RNase H (Enzymatics) at37° C. for 1 hour. The RCA primer is then hybridized to the sample at60° C. for 15 min and washed. For rolling circle amplification, 100 Uphi29 DNA polymerase (Enzymatics), 250 uM dNTP and 40 uM aminoallyl dNTPare added to the sample and incubated at 30° C. overnight. The sample isthen washed using PBS, and cross-linked using 100 uM BS(PEG)9 in PBS for1 hour, followed by 1M Tris treatment for 15 min. For the DNA amplicondetection, 1 uM fluorescently label oligonucleotides are diluted in2×SSC and hybridized to the matrix containing the DNA amplicons at 60°C. and washed. Imaging is done using Leica epifluorescence microscopeusing a 10× objective in four color channels (FITC, Cy3, Texas Red andCy5) in a tiled scan mode (20 by 15 separate images). The images arethen stitched together during the image acquisition and visualized usingImaris Bitplane software.

As shown in FIG. 2, mouse brain sections were obtained and DNA/RNA wasamplified in situ. The DNA/RNA was copolymerized into a matrix materialin situ, and individual amplicons were interrogated/hybridized withfluorescent oligonucleotides and imaged. When viewed under much highermagnification, individual amplicons can be imaged using confocalmicroscopy even in these thick biological specimens. This allows one tofind out where different DNA/RNA molecules reside, how they arecompartmentalized among different cell types and morphologies and howtheir representation changes over time in developing tissues. Thesimilar concept can be used for many other specimens in both natural andsynthetic materials, as long as they can be co-polymerized and/orencapsulated by the DNA amplicons.

Example IV Crosslinking of Amplicons

A 50-base oligonucleotide is phosphorylated at the 5′ end usingpolynucleotide kinase in the T4 ligase buffer for 15 min. The reactionmixture is incubated with CircLigase mixture at 60° C. for 1 hour togenerate circular templates for testing. The RCA primer (18 bases) isthen hybridized to the circular template in solution and a dilutedtemplate:primer mixture is used for rolling circle amplification. TheRCA reaction solution contained 0, 0.1 uM, 1 uM or 10 uM aminoallyl dUTPin addition to the normal dNTP. The reaction mixture was then loadedonto an 1% agarose gel and visualized using SYBR safe dyes.

The incorporation of aminoallyl dUTP that is later cross-linked to eachother and to the amine exposing substrate still allows for reversetranscription using M-MuLV reverse transcriptase and rolling circleamplification using Phi29 DNA polymerase, albeit at a reduced rate in aconcentration dependent manner. Increasing amounts of aminoallyl dUTPwere added as a competitor to dTTP present in the amplification mixturein solution. The rolling circle amplicons are single stranded DNA whichare highly folded. As shown in FIG. 3A, these structures run as a singlelarge band around ˜10-15-kb on an 1% agarose gel.

Example V The Incorporation of Aminoallyl dUTP Leads to Slightly SmallerDiameter of the Average DNA Amplicon Size

A 50-base oligonucleotide is phosphorylated at the 5′ end usingpolynucleotide kinase in the T4 ligase buffer for 15 min. The reactionmixture is incubated with CircLigase mixture at 60° C. for 1 hour togenerate circular templates for testing. The RCA primer (18 bases) isthen hybridized to the circular template in solution and a dilutedtemplate:primer mixture is used for rolling circle amplification. TheRCA reaction solution contained aminoallyl dUTP and normal dNTP atvarying ratios (1:50 to 1:50,000). After 8 hours of RCA, the reactionmixture was diluted in PBS and bound to amino-silane treated coverglass.The bound RCA amplicons were then visualized by staining it with SYBRsafe and imaging it using an epifluorescence microscope (63× objective).The images were then processed using Imaris Bitplane to identifyindividual amplicons and measure the average diameter of each spot.

The incorporation of aminoallyl dUTP leads to slightly smaller diameterof the average DNA amplicon size. The circular cDNA template was usedfor rolling circle amplification, during which a range of aminoallyldUTP was added. The amplicon mixture in solution was then arrayed on aglass surface and hybridized to a common fluorescent probe sequence.Since aminoallyl dUTP has a single positive charge, the increasingincorporation of aminoallyl dUTP led to a reduction in the overallnegative charge, making each DNA amplicon slightly more compact. Asshown in FIG. 3B, the ratio shown in the graph legend represents themolar ratio of aminoallyl dUTP to dTTP during the amplification step.

Example VI Aminoallyl dUTP Cross-Linking Preserves DNA Amplicons

A 50-base oligonucleotide is phosphorylated at the 5′ end usingpolynucleotide kinase in the T4 ligase buffer for 15 min. The reactionmixture is incubated with CircLigase mixture at 60° C. for 1 hour togenerate circular templates for testing. The RCA primer (18 bases) isthen hybridized to the circular template in solution and a dilutedtemplate:primer mixture is used for rolling circle amplification with orwithout aminoallyl dUTP. After 8 hours of RCA, the reaction mixture wasdiluted in PBS and bound to amino-silane treated coverglass. The boundRCA amplicons were then cross-linked with BS(PEG)9. They were thenwashed using a continuous stream of 2×SSC wash solution for 1 min,stained with SYBR safe and imaged using an epifluorescence microscope(63× objective).

The DNA amplicons generated in solution are arrayed on a glass surfaceand cross-linked via the aminoallyl moiety. They were then exposed to aconstant flow of distilled water running across its surface with andwith the cross-linker chemistry for 5 min at room temperature and thenimaged after SYBR Gold staining. As shown in FIG. 3C, the DNA ampliconsthat were not cross-linked stretched out as a result of high shearstress for about 5 minutes. The DNA amplicons cross-linked withaminoallyl dUTP were morphologically preserved after high shear stressfor 5 minutes.

Example VII DNA Amplicons in Human Fibroblasts are Structurally andChemically Stable

Human primary fibroblasts are grown on a 1.5 cover slip. They are fixedusing 4% formaldehyde in PBS for 15 min, followed by three washes of 70%ethanol. The reverse transcription mixture containing 1 uM randomhexamer or 0.1 uM polydT(18)V primer with additional adapter sequences,250 uM dNTP, 40 uM aminoallyl dUTP, 20 U RNase inhibitor and 100 UM-MuLV reverse transcriptase are then added to the fixed cells andincubated overnight at 37° C. The sample is then washed using PBS, andcross-linked using 100 uM BS(PEG)9 in PBS for 1 hour, followed by 1MTris treatment for 15 min. The circularization mixture containing 25 UCircLigase, 1 mM MnCl and 1 M Betain is added, and the sample isincubated at 60° C. The residual RNA is degraded using a mixture ofRNase cocktail and RNase H. The RCA primer is then hybridized to thesample at 60° C. for 15 min. For rolling circle amplification, 100 Uphi29 DNA polymerase, 250 uM dNTP and 40 uM aminoallyl dNTP are added tothe sample and incubated at 30° C. overnight. The sample is then washedusing PBS, and cross-linked using 100 uM BS(PEG)9 in PBS for 1 hour,followed by 1M Tris treatment for 15 min. For the DNA amplicondetection, 1 uM fluorescently labeled oligonucleotides are diluted in2×SSC and hybridized to the matrix containing the DNA amplicons at 60°C. and washed. Imaging is done using Leica SP5 scanning confocalmicroscope using a 63× objectives. The fluorescent oligonucleotides arestripped off using 80% formamide heated to 80° C. The sample is thendried and stored at 4° C. from July 2011 to March 2012. The sample wasrehydrated in PBS, and rehybridized to the fluorescently labeledoligonucleotides and imaged. The second image was obtained using anepifluorescence microscope.

The DNA amplicons are structurally and chemically stable over a longperiod of time once cross-linked. As shown in FIG. 4A, the DNA ampliconspreserved as a three dimensional matrix in human fibroblasts can beinterrogated using fluorescent primers and stored in phosphate bufferedsolution for up to a year and re-interrogated without losing theirstructural or sequence information. The different image quality herereflects the difference between confocal microscopy vs. epifluorescencemicroscopy, not the sample quality.

Example VIII A DNA Amplicon Matrix within a Cell is Structurally andChemically Stable

Human primary fibroblasts are grown on a 1.5 cover slip. They are fixedusing 4% formaldehyde in PBS for 15 min, followed by three washes of 70%ethanol. The reverse transcription mixture containing 1 uM randomhexamer or 0.1 uM polydT(18)V primer with additional adapter sequences,250 uM dNTP, 40 uM aminoallyl dUTP, 20 U RNase inhibitor and 100 UM-MuLV reverse transcriptase are then added to the fixed cells andincubated overnight at 37° C. The sample is then washed using PBS, andcross-linked using 100 uM BS(PEG)9 in PBS for 1 hour, followed by 1MTris treatment for 15 min. The circularization mixture containing 25 UCircLigase, 1 mM MnCl and 1 M Betain is added, and the sample isincubated at 60° C. The residual RNA is degraded using a mixture ofRNase cocktail and RNase H. The RCA primer is then hybridized to thesample at 60° C. for 15 min. For rolling circle amplification, 100 Uphi29 DNA polymerase, 250 uM dNTP and 40 uM aminoallyl dNTP are added tothe sample and incubated at 30° C. overnight. The sample is then washedusing PBS, and cross-linked using 100 uM BS(PEG)9 in PBS for 1 hour,followed by 1M Tris treatment for 15 min. For the DNA amplicondetection, 1 uM fluorescently labeled oligonucleotides are diluted in2×SSC and hybridized to the matrix containing the DNA amplicons at 60°C. and washed. Imaging is done using Leica SP5 scanning confocalmicroscope using a 63× objectives. The fluorescent oligonucleotides arestripped off using 80% formamide heated to 80° C. The sample is thenwashed with distilled waterand rehybridized to the fluorescently labeledoligonucleotides and imaged.

As shown in FIG. 4B, the DNA amplicon matrix inside the cell can bestripped using harsh chemical agents (i.e. 0.1N NaOH, 80% formamide) andheated up to 95° C. for a prolonged period of time without losing theirstructural integrity or definition.

Example IX A DNA Amplicon Matrix within a Cell is Structurally andChemically Stable

Human iPS cells are grown on a 1.5 cover slip. They are fixed using 4%formaldehyde in PBS for 15 min, followed by three washes of 70% ethanol.The reverse transcription mixture containing 1 uM random hexamer or 0.1uM polydT(18)V primer with additional adapter sequences, 250 uM dNTP, 40uM aminoallyl dUTP, 20 U RNase inhibitor and 100 U M-MuLV reversetranscriptase are then added to the fixed cells and incubated overnightat 37° C. The sample is then washed using PBS, and cross-linked using100 uM BS(PEG)9 in PBS for 1 hour, followed by 1M Tris treatment for 15min. The circularization mixture containing 25 U CircLigase, 1 mM MnCland 1 M Betain is added, and the sample is incubated at 60° C. Theresidual RNA is degraded using a mixture of RNase cocktail and RNase H.The RCA primer is then hybridized to the sample at 60° C. for 15 min.For rolling circle amplification, 100 U phi29 DNA polymerase, 250 uMdNTP and 40 uM aminoallyl dNTP are added to the sample and incubated at30° C. overnight. The sample is then washed using PBS, and cross-linkedusing 100 uM BS(PEG)9 in PBS for 1 hour, followed by 1M Tris treatmentfor 15 min. For the DNA amplicon detection, 1 uM fluorescently labeledoligonucleotides are diluted in 2×SSC and hybridized to the matrixcontaining the DNA amplicons at 60° C. and washed three times using2×SSC. After imaging, the fluorescent oligonucleotides are stripped offusing 80% formamide heated to 80° C. The sample is then washed withdistilled water and rehybridized to the fluorescently labeledoligonucleotides. This cycle is repeated sixty times (5 minutes percycle). The multiple images were then aligned and processed using MatLabto identify a region of interest. Up to twenty single cells andamplicons within the cells were chosen and compared to the cell-freeregion for determining the signal to noise ratio after eachhybridization and stripping cycle. The sample was then used for 12cycles of sequencing by ligation. After sequencing, the 12 image stacks(19 optical sections per field) were analyzed on Imaris Bitplane andindividual DNA amplicons were tracked over the whole sequencing run.Only those amplicons that were identified in all 12 cycles wereanalyzed.

As shown in FIG. 4C, the sample can be cycled through more than 50heating, cooling, enzymatic and chemical reactions without any changesin the signal to noise ratio. The high absolute signal intensity herewas due to insufficient probe washing in the initial cycles. When theindividual DNA amplicons in the matrix was imaged in three dimensionsusing confocal microscopy and tracked over 12 cycles, one measure therelative displacement of each amplicon over time. Despite numerousthermal, chemical and enzymatic manipulations, the mean displacement ofeach amplicon was ˜500-nm in both lateral and axial dimensions, whichwas about the diameter of each amplicon. An example image of theanalysis is shown in the right panel, in which the line representing thedisplacement is shown in different colors according to their cyclenumber.

Example X DNA Amplicons Embedded within a Cross-Linked Matrix in a Cellare Imaged

Human iPS cells are grown on a 1.5 cover slip. They are fixed using 4%formaldehyde in PBS for 15 min, followed by three washes of 70% ethanol.The reverse transcription mixture containing 1 uM random hexamer or 0.1uM polydT(18)V primer with additional adapter sequences, 250 uM dNTP, 40uM aminoallyl dUTP, 20 U RNase inhibitor and 100 U M-MuLV reversetranscriptase are then added to the fixed cells and incubated overnightat 37° C. The sample is then washed using PBS, and cross-linked using100 uM BS(PEG)9 in PBS for 1 hour, followed by 1M Tris treatment for 15min. The circularization mixture containing 25 U CircLigase, 1 mM MnCland 1 M Betain is added, and the sample is incubated at 60° C. Theresidual RNA is degraded using a mixture of RNase cocktail and RNase H.The RCA primer is then hybridized to the sample at 60° C. for 15 min.For rolling circle amplification, 100 U phi29 DNA polymerase, 250 uMdNTP and 40 uM aminoallyl dNTP are added to the sample and incubated at30° C. overnight. The sample is then washed using PBS, and cross-linkedusing 100 uM BS(PEG)9 in PBS for 1 hour, followed by 1M Tris treatmentfor 15 min. For the DNA amplicon detection, 1 uM fluorescently labeledoligonucleotides are diluted in 2×SSC and hybridized to the matrixcontaining the DNA amplicons at 60° C. and washed three times using2×SSC. Leica SP5 scanning confocal microscope with 63× objective is usedand scanning optical zoom of 5× is used. The line scan was repeatedthree times and averaged to generate a high quality image.

FIG. 5A is an image of the DNA amplicons (derived from reversetranscription of the cytoplasmic and the nuclear RNA) embedded withinthe cross-linked matrix inside human induced pluripotent stem cells.Individual amplicons are too tightly packed to visualize discreteamplicons, given the optical diffraction limitation in microscopy. Butvarious subcellular compartments where the RNA is not expected to bepresent (i.e. nm: nuclear membrane, pm: plasma membrane) show darkstaining, whereas the nucleus (Nu) and the cytoplasm (Cy) show a highdensity of the amplicons. The distribution of the cellular RNA showunique patterns from cell to cell (1st panel vs. 2nd panel) and from onecell cycle phase to another (1st panel vs. 3rd panel). These resultsshow that the DNA amplicons can be immobilized, amplified andinterrogated in a manner to reflect their original spatial information.

Example XI DNA Amplicons Embedded within a Cross-Linked Matrix in a Cellare Sequenced

Human iPS cells are grown on a 1.5 cover slip. They are fixed using 4%formaldehyde in PBS for 15 min, followed by three washes of 70% ethanol.The reverse transcription mixture containing 1 uM random hexamer or 0.1uM polydT(18)V primer with additional adapter sequences, 250 uM dNTP, 40uM aminoallyl dUTP, 20 U RNase inhibitor and 100 U M-MuLV reversetranscriptase are then added to the fixed cells and incubated overnightat 37° C. The sample is then washed using PBS, and cross-linked using100 uM BS(PEG)9 in PBS for 1 hour, followed by 1M Tris treatment for 15min. The circularization mixture containing 25 U CircLigase, 1 mM MnCland 1 M Betain is added, and the sample is incubated at 60° C. Theresidual RNA is degraded using a mixture of RNase cocktail and RNase H.The RCA primer is then hybridized to the sample at 60° C. for 15 min.For rolling circle amplification, 100 U phi29 DNA polymerase, 250 uMdNTP and 40 uM aminoallyl dNTP are added to the sample and incubated at30° C. overnight. The sample is then washed using PBS, and cross-linkedusing 100 uM BS(PEG)9 in PBS for 1 hour, followed by 1M Tris treatmentfor 15 min. The sequencing primer is designed with different 3′ endsthat that each primer can detect only ¼th of the amplicons. If differentdinucleotides are added to the 3′ ends of the primer, each primer candetect only 1/16th of the amplicons. A chosen sequencing primer in 2×SSCis hybridized to the sample at 60° C. for 15 minutes and washed. Aligation mixture containing 10 U T4 DNA ligase, ligation buffer and 1 uMfluorescently labeled nonamers (a pool containing A, G, C or T at fixedpositions and labeled with FITC, Cy3, Texas Red or Cy5, respectively) isadded and incubated for 50 min at room temperature. After washing threetimes with 2×SSC, the cell is imaged on Leica SP5 scanning confocalmicroscope using four color channels. After imaging, the probe complexis stripped using 80% formamide and washed with distilled water. Thesequencing by ligation step is repeated using a different nonamer setinterrogating the next sequence.

Using selective sequencing primers, only a subset of the total ampliconscan be sequenced for better spatial resolution. The left panel shows asubset of randomly primed cDNA amplicons being sequenced on a confocalmicroscope. The right panel shows GAPDH cDNA amplicons being sequencedover time using confocal microscopy. Only a single optical section isshown here. The axial dimension represents time or sequencing cyclesteps.

Example XII Circular DNA is Cross-Linked or Co-Polymerized into a Matrixand Amplified

As shown in FIG. 6, circular DNA, including cDNA, is first modified toincorporate a given cross-linker chemistry (i.e. aminoallyl, thiol,biotin) using modified dUTP that competes with natural dTTP. Thecircular DNA is then cross-linked and/or co-polymerized within a threedimensional container (i.e. cell), conforming the shape and the size ofthe container. Uncross-linked molecules are then washed away, and onethen performs rolling circle amplification, followed by imaging (i.e.sequencing). The density, the size and the signal strength can becontrolled by varying the template size, the amplification time and thedetection primer sequence. The DNA amplicons can be made into an ordered3D matrix in a suitable scaffold material with addressable primers thatcan serve as amplification primers.

Example XIII References

Each reference is incorporated herein by reference in its entirety forall purposes.

-   Drmanac, R., Sparks, A. B., Callow, M. J., Halpern, A. L., Burns, N.    L., Kermani, B. G., Carnevali, P., Nazarenko, I., Nilsen, G. B.,    Yeung, G., et al. (2010). Human genome sequencing using unchained    base reads on self-assembling DNA nanoarrays. Science 327, 78-81.-   Islam, S., Kjallquist, U., Moliner, A., Zajac, P., Fan, J. B.,    Lonnerberg, P., and Linnarsson, S. (2011). Characterization of the    single-cell transcriptional landscape by highly multiplex RNA-seq.    Genome Res 21, 1160-1167.-   Larsson, C., Grundberg, I., Söderberg, O., and Nilsson, M. (2010).    In situ detection and genotyping of individual mRNA molecules.    Nature methods 7, 395-397.-   Larsson, C., Koch, J., Nygren, A., Janssen, G., Raap, A. K.,    Landegren, U., and Nilsson, M. (2004). In situ genotyping individual    DNA molecules by target-primed rolling-circle amplification of    padlock probes. Nature methods 1, 227-232.-   Shendure, J., Porreca, G. J., Reppas, N. B., Lin, X., McCutcheon, J.    P., Rosenbaum, A. M., Wang, M. D., Zhang, K., Mitra, R. D., and    Church, G. M. (2005). Accurate multiplex polony sequencing of an    evolved bacterial genome. Science 309, 1728-1732.

What is claimed is:
 1. A method for analysis, comprising: (a) providinga biological sample comprising a plurality of cells, wherein a cell ofsaid plurality of cells comprises a plurality of cellular nucleic acids;(b) generating a three-dimensional matrix comprising cellular nucleicacids of said plurality of cellular nucleic acids or derivatives of saidcellular nucleic acids, wherein said cellular nucleic acids orderivatives of said cellular nucleic acids have a three-dimensionalposition in said three-dimensional matrix; and (c) detecting signalsfrom said cellular nucleic acids or derivatives of said cellular nucleicacids to identify said three-dimensional position in saidthree-dimensional matrix.
 2. The method of claim 1, further comprising,prior to (b), permeabilizing said cell.
 3. The method of claim 2,wherein (b) comprises contacting said cell with a matrix-formingmaterial and using said matrix-forming material to form saidthree-dimensional matrix.
 4. The method of claim 3, wherein saidthree-dimensional matrix is a cross-linked or polymer matrix.
 5. Themethod of claim 3, wherein said three-dimensional matrix comprisespolyacrylamide, cellulose, alginate, polyamide, cross-linked agarose,cross-linked dextran, or cross-linked polyethylene glycol.
 6. The methodof claim 3, wherein (b) further comprises exposing said matrix-formingmaterial to a polymerization inducing catalyst, ultraviolet (UV) light,or a functional cross-linker.
 7. The method of claim 3, wherein saidmatrix-forming material comprises a polymer, wherein saidthree-dimensional polymer matrix is generated at least in part bycrosslinking said polymer.
 8. The method of claim 3, wherein saidmatrix-forming material comprises a plurality of monomers, and whereinsaid three-dimensional matrix is generated at least in part bypolymerizing said plurality of monomers.
 9. The method of claim 1,further comprising, prior to (b), generating a plurality of ampliconsfrom said cellular nucleic acids.
 10. The method of claim 9, whereinsaid derivatives of said cellular nucleic acids comprise amplicons ofsaid plurality of amplicons and (b) comprises generating saidthree-dimensional matrix comprising said amplicons, wherein saidamplicons have said three-dimensional position in said three-dimensionalmatrix.
 11. The method of claim 10, wherein (b) comprises covalentlyattaching said amplicons to said three-dimensional matrix therebysubstantially retaining said three-dimensional position of saidamplicons in said three-dimensional matrix.
 12. The method of claim 10,wherein said amplicons comprise a functional moiety and wherein saidamplicons are covalently attached to said three-dimensional matrix viasaid functional moiety.
 13. The method of claim 12, wherein saidfunctional moiety comprises an amine, an acrydite, an alkyne, a biotin,an azide, or a thiol.
 14. The method of claim 12, wherein said ampliconscomprising said functional moiety are generated by performing a nucleicacid amplification reaction in the presence of a modified nucleotidecomprising said functional moiety such that said modified nucleotide isincorporated into said amplicons.
 15. The method of claim 14, whereinsaid modified nucleotide comprises uracil.
 16. The method of claim 12,wherein said functional moiety is covalently cross-linked to saidthree-dimensional matrix to covalently attach said amplicons to saidthree-dimensional matrix.
 17. The method of claim 16, further comprisingusing a cross-linker to covalently attach said amplicons to saidthree-dimensional matrix, wherein said cross-linker comprises a reactivegroup that reacts with said functional moiety.
 18. The method of claim17, wherein said reactive group comprises an imidoester, a succinimideester, a maleimide, a carbodiimide, or a phenyl azide.
 19. The method ofclaim 17, wherein said cross-linker further comprises a spacer.
 20. Themethod of claim 19, wherein said spacer comprises polyethylene glycol, acarbon backbone, or a photo-cleavable moiety.
 21. The method of claim12, wherein said functional moiety is co-polymerized with matrix-formingmaterial to covalently attach said amplicons to said three-dimensionalmatrix.
 22. The method of claim 21, wherein said functional moietycomprises acrydite and wherein said matrix forming material comprises anacrylamide.
 23. The method of claim 10, wherein said amplicons arelabelled with detectable labels, and wherein (c) comprises detectingsignals from said detectable labels.
 24. The method of claim 23, whereinsaid detectable labels are fluorescent labels.
 25. The method of claim23, wherein detecting signals from said detectable labels comprisesimaging.
 26. The method of claim 23, wherein said signals are detectedin a plurality of optical sections to identify said three-dimensionalposition in said three-dimensional matrix.
 27. The method of claim 23,further comprising generating a visualization comprising athree-dimensional reconstruction of said three-dimensional position insaid three-dimensional matrix.
 28. The method of claim 2, furthercomprising, prior to permeabilizing said cell, using a fixative tosubstantially fix said cellular nucleic acids.
 29. The method of claim10, wherein (c) comprises sequencing said amplicons.
 30. A method foranalysis, comprising: (a) providing a biological sample comprising aplurality of cells, wherein a cell of said plurality of cells comprisesa plurality of cellular nucleic acids; (b) generating a plurality ofamplicons from said plurality of cellular nucleic acids; (c) generatinga three-dimensional matrix comprising amplicons of said plurality ofamplicons, wherein said amplicons are covalently attached to saidthree-dimensional matrix, and wherein said amplicons have athree-dimensional position in said three-dimensional matrix; and (d)hybridizing fluorescently labeled oligonucleotides to said amplicons andimaging said amplicons to identify said three-dimensional position insaid three-dimensional matrix.
 31. The method of claim 29, wherein saidamplicons are generated through rolling circle amplification ofcircularized probes.